Expression of a biologically active antiviral antibody using a sindbis virus vector system.
Monoclonal antibodies to the Sindbis virus E2 envelope glycoprotein protect mice against lethal encephalitis and mediate viral clearance from neurons. To facilitate structure-function analyses of anti-E2 mAbs, we developed an expression system that can be used for the construction of genetically engineered anti-E2 mAbs. We constructed recombinant Sindbis/immunoglobulin gene chimeric viruses that express heavy and light chains of an anti-E2 monoclonal antibody, R6. We used a PCR-based strategy to clone the entire rearranged heavy and light chain genes from R6 hybridoma cell cDNA into a double subgenomic Sindbis virus vector. The recombinant viruses, SIN/R6L and SIN/R6H, were generated by transfecting BHK-21 cells with in vitro transcribed RNA from Sindbis virus/R6 light chain and Sindbis virus/R6 heavy chain cDNA clones, respectively. Twelve hours after co-infection of BHK cells with SIN/R6L and SIN/R6H, the tissue culture supernatant contained up to 1.4 mg/ml of recombinant R6 IgG. The heavy and light chains of recombinant R6 were associated as judged by co-purification on protein A/G sepharose and co-electrophoresis of non-reduced proteins. The ELISA reactivity to Sindbis virus antigen was similar for recombinant R6 and R6 purified from ascites fluid. Furthermore, the in vivo biologic activity of recombinant R6 was similar to that of R6 purified from ascites; recombinant R6 treatment completely protected Balb/cJ mice from paralysis and death due to infection with neuroadapted Sindbis virus and also resulted in the clearance of infectious virus from the brains of immunodeficient scid mice persistently infected with wild-type Sindbis virus. Thus, the co-infection of BHK cells with SIN/R6L and SIN/R6H leads to the expression, assembly, and secretion of a biologically active recombinant antiviral antibody. Our results suggest that the Sindbis virus vector system is a simple and powerful tool for the production of functional, genetically engineered antibodies.
Liang
X H
XH
Department of Medicine, Columbia University College of Physicians & Surgeons, New York, NY 10032, USA.
Jiang
H H
HH
Levine
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000627:therapeutic use; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004675:Encephalitis, Tick-Borne / Q000517:prevention & control; D005818:Genetic Engineering; D005822:Genetic Vectors; D007116:Immunization, Passive; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000627:therapeutic use; D015183:Restriction Mapping; D012845:Sindbis Virus / Q000276:immunology*; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C064950:glycoprotein E2, Sindbis virus